A pancreatic cancer organoid culture medium and a pancreatic cancer organoid culture method

Abstract

The application provides a pancreatic cancer organoid culture medium and a pancreatic cancer organoid culture method, and belongs to the technical field of organoid culture.The application adopts specific types of base culture medium, additives, cytokines and inhibitors to obtain a pancreatic cancer organoid culture medium, so that the problem of small product size and low activity in the prior art pancreatic cancer organoid culture can be solved.The pancreatic cancer organoid culture medium can significantly improve the size and activity of the obtained organoid, and the culture method provided by the application can obtain a faster cell proliferation speed, so that the efficient production of pancreatic cancer organoids can be realized in industry, and a better material is provided for in-vitro tumor biology and treatment-related research, and has a wide application prospect.

Description

Technical Field

[0001] This invention relates to the field of organoid culture technology, and more particularly to a pancreatic cancer organoid culture medium and a method for culturing pancreatic cancer organoids. Background Technology

[0002] Tumor organoids are three-dimensional models created from fresh tumor tissue derived from patients using an in vitro three-dimensional (3D) cell culture system. They are highly similar to the characteristics of the patient's tumor. Therefore, "organoid" means similar to a tissue or organ. They can serve as a substitute for the patient and become an in vitro model for guiding personalized medicine in clinical practice.

[0003] In 2009, researchers in this field cultivated the first miniature intestinal organoid using adult stem cells derived from the mouse intestine, sparking a surge of interest in organoid research. In 2011, researchers pioneered the establishment of tumor organoids using organoid technology. They cultured human small intestine and colon organoids by adjusting the culture environment based on the culture conditions of mouse colonic crypts, and then further optimized and adjusted them to establish tumor organoid models from colonic adenomas, adenocarcinomas, and Barrett's esophagus.

[0004] The establishment of tumor organoid models derived from cancer patients is considered another major breakthrough in organoid culture technology. Tumor organoids are cell clusters formed from tumor-specific stem cells in tumor tissue through three-dimensional tissue culture. They can simulate the characteristics and heterogeneity of tumor cells in vivo. The establishment of this technology has provided a reliable model for cancer research and treatment, especially opening up new horizons for personalized cancer therapy. Currently, researchers have successfully generated many tumor organoids by optimizing various culture conditions, including those for colon cancer, prostate cancer, gastric cancer, breast and pancreatic cancer, endometrial / ovarian cancer, uterine carcinosarcoma, urothelial carcinoma, and renal cancer. Compared with traditional tumor research models, tumor organoids can simulate the structure and function of tumors in vivo to the greatest extent. They can intuitively show the tumor growth process and reflect individual patient differences. It is an intuitive, reliable, efficient, and ethically sound organ-level research system, making it more suitable for in vitro tumor biology and treatment-related research.

[0005] Chinese patent CN116555181A discloses a pancreatic cancer organoid, culture medium, and culture method. The pancreatic cancer organoid culture medium is characterized by comprising a basic culture and specific additive factors. The pancreatic cancer organoid culture medium provided by this invention contains the minimum components required for pancreatic cancer organoid culture. These components do not require bovine serum albumin, the most common component in cell culture, thus saving costs and reducing cytotoxicity and inhibitors introduced by free radical styrene (FBS). The pancreatic cancer organoid culture medium provided by this invention is suitable for the culture of pancreatic cancer organoids. The various components of the organoid culture medium work synergistically, enabling the cultured pancreatic cancer organoids to maintain the morphology, structure, and genetic characteristics of the primary tissue.

[0006] However, the above culture method can only obtain pancreatic cancer organoids with a size of about 200 μm, and their activity needs to be improved. Summary of the Invention

[0007] The purpose of this invention is to provide a pancreatic cancer organoid culture medium and a method for culturing pancreatic cancer organoids, which can obtain larger pancreatic cancer organoids with better activity.

[0008] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0009] This invention provides a pancreatic cancer organoid culture medium, comprising the following components:

[0010] Basic culture medium, additives, cytokines, and inhibitors;

[0011] The cytokines include R-Spondin 1, Noggin, epidermal growth factor, and B27 supplement.

[0012] Preferably, the final concentration of R-Spondin 1 in the pancreatic cancer organoid culture medium is 50–150 ng / ml;

[0013] The final concentration of Noggin is 50–150 ng / ml;

[0014] The final concentration of the epidermal growth factor is 50–150 ng / ml;

[0015] The B27 supplement is used at a dilution factor of 1X.

[0016] Preferably, the cytokines further include fibroblast growth factor 10;

[0017] Preferably, the final concentration of the fibroblast growth factor 10 is 30-80 ng / ml.

[0018] Preferably, the inhibitors include N-acetylcysteine, nicotinamide, A83-01, and Y-27632;

[0019] Preferably, the final concentration of N-acetylcysteine ​​in the pancreatic cancer organoid culture medium is 0.5–2 mM;

[0020] The final concentration of the nicotinamide is 5–20 mM;

[0021] The final concentration of A83-01 is 0.3–0.8 μM;

[0022] The final concentration of Y-27632 is 5–20 μM.

[0023] Preferably, the inhibitor further includes gastrin;

[0024] Preferably, the final concentration of gastrin in the pancreatic cancer organoid culture medium is 5–20 nM.

[0025] Preferably, the additive includes one or more of 4-hydroxyethylpiperazine ethanesulfonic acid, antibiotics, and L-alanyl-L-glutamine;

[0026] Preferably, in the pancreatic cancer organoid culture medium, the final concentration of 4-hydroxyethylpiperazine ethanesulfonic acid is 5-20 mM;

[0027] The final concentration of the antibiotic is 1X;

[0028] The final concentration of the L-alanyl-L-glutamine is 2 mM.

[0029] Preferably, the basal culture medium is Advanced DMEM / F12.

[0030] This invention also provides a method for culturing pancreatic cancer organoids, comprising the following steps:

[0031] Pancreatic cancer tissue was mixed with digestive juices and digested to obtain digestible tissue.

[0032] The digestive tissue was filtered using a cell filter to obtain a precipitate;

[0033] The precipitate was purified to obtain cells;

[0034] Cells were seeded into the above-mentioned pancreatic cancer organoid culture medium for organoid growth culture to obtain pancreatic cancer organoids.

[0035] Preferably, the digestive fluid contains type 2 collagenase and neutral protease.

[0036] Preferably, the final concentration of type 2 collagenase in the digestive fluid is 1–3 mg / ml;

[0037] The final concentration of neutral protease in the digestive fluid is 0.01–0.03 U / ml;

[0038] And / or, the digestion process takes 1 to 2 hours.

[0039] The beneficial effects of this invention are:

[0040] This invention provides a pancreatic cancer organoid culture medium by combining specific types of basal culture media, additives, cytokines, and inhibitors. Using this medium to culture pancreatic cancer organoids significantly increases the size and activity of the resulting organoids. Furthermore, the culture method provided by this invention enables faster cell proliferation. This medium holds promise for achieving efficient production of pancreatic cancer organoids in industry, providing superior materials for in vitro tumor biology and therapeutic research, and demonstrating broad application prospects. Attached Figure Description

[0041] Figure 1 These are images showing the cell culture status on the first and third days after cell seeding in Example 1;

[0042] Figure 2 These are images showing the cell culture status on the first and third days after cell seeding in Example 2;

[0043] Figure 3 This is a diagram of the final pancreatic cancer organoid obtained in Example 1. Detailed Implementation

[0044] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0045] Example 1

[0046] Preparation of culture medium:

[0047] Table 1. Culture medium formulation for Example 1

[0048]

[0049] Cultivate:

[0050] Collect fresh pancreatic cancer tissue samples and immediately place them in a tissue preservation solution at 4°C.

[0051] Add ice-cold PBS containing 1% penicillin antibody to a 35 mm dish. Remove the pancreatic cancer tissue sample, discard normal tissue, fat, blood clots, and necrotic areas, and cut the pancreatic cancer sample into 1-2 mm pieces using surgical scissors or a scalpel. 3 Small fragments.

[0052] Transfer tissue fragments into 10ml centrifuge tubes and allow the tissues to settle under gravity. Remove the supernatant and wash twice with ice-cold PBS containing 1% penicillin and antibiotics.

[0053] Add 5 ml of pancreatic cancer organoid digestion solution (2 mg / ml type 2 collagenase, 1 U neutral protease) according to the tissue volume, seal with sealing film, and digest on a shaker at 37°C for 1.5 h, shaking and mixing once every 30 min.

[0054] After digestion is complete, the tissue is further digested by blowing and purging 10 times with a 1ml pipette tip, then filtered using a 100μm cell filter, centrifuged at 300g for 5 minutes, and the supernatant is removed.

[0055] Resuspend in 5 ml adDMEM / F12+++ (containing antibiotics, glutamine, and HEPES), centrifuge at 300 g for 5 min, and remove the supernatant.

[0056] Based on the amount of precipitate, add the appropriate volume of pancreatic cancer organoid culture medium and matrix gel 1:1 to resuspend.

[0057] Remove the 24-well plate that was placed 2 hours in advance from the 37℃ incubator, add 50 μl of the mixture to each well, place it in the 37℃ incubator for 5 minutes, and then invert it to solidify for 20 minutes.

[0058] After solidification, 500 μl of pancreatic cancer organoid culture medium was added to a 24-well plate and placed in a cell culture incubator for culture. The growth of the organoids was observed daily, and the culture medium was changed every 2-3 days.

[0059] Cultivation photos such as Figure 1 As shown, the survival rate was 90% on the first day, the average size of the organoids was 250 μm on the third day, and the final product was obtained on the seventh day, with an average organoid size of 600 μm. Figure 3 As shown.

[0060] Example 2

[0061] Table 2. Culture medium formulation for Example 2

[0062]

[0063]

[0064] The culture program is the same as in Example 1.

[0065] Cultivation photos such as Figure 2 As shown, the survival rate on the first day was 50%, the average size of the organoids on the third day was 200 μm, and the final product was obtained on the seventh day with an average size of 300 μm.

[0066] The results showed that the organoids in Example 1 of the present invention grew very well on the first day, while the organoids in Example 2 exhibited apoptosis on the first day. The organoids in Example 1 grew faster and were larger on the first day, while the organoids in Example 2 grew slower, but the final size of the cultured organoids was still better than that of the organoids in the prior art (around 100 μm).

[0067] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A culture medium for pancreatic cancer organoids, characterized in that, Includes the following components: Basic culture medium, additives, cytokines, inhibitors, and gastrin; The cytokines include R-Spondin 1, Noggin, epidermal growth factor, and B27 supplement; In the pancreatic cancer organoid culture medium, the final concentration of R-Spondin 1 is 50~150 ng / ml; The final concentration of Noggin is 50~150 ng / ml; The final concentration of the epidermal growth factor is 50~150 ng / ml; The B27 supplement is used at a dilution factor of 1X. The cytokines also include fibroblast growth factor 10; The final concentration of the fibroblast growth factor 10 is 30~80 ng / ml; The inhibitors include N-acetylcysteine, nicotinamide, A83-01, and Y-27632; In the pancreatic cancer organoid culture medium, the final concentration of N-acetylcysteine ​​is 0.5~2 mM; The final concentration of the nicotinamide is 5~20 mM; The final concentration of A83-01 is 0.3~0.8µM; The final concentration of Y-27632 is 5~20 µM; The final concentration of gastrin is 5-20 nM; The additives include one or more of 4-hydroxyethylpiperazine ethanesulfonic acid, antibiotics, and L-alanyl-L-glutamine; In the pancreatic cancer organoid culture medium, the final concentration of 4-hydroxyethylpiperazine ethanesulfonic acid is 5~20mM; The final concentration of the antibiotic is 1X; The final concentration of the L-alanyl-L-glutamine is 2 mM.

2. The pancreatic cancer organoid culture medium according to claim 1, characterized in that, The basic culture medium is Advanced DMEM / F12.

3. A method for culturing pancreatic cancer organoids, characterized in that, Includes the following steps: Pancreatic cancer tissue was mixed with digestive juices and digested to obtain digestible tissue. The digestive tissue was filtered using a cell filter to obtain a precipitate; The precipitate was purified to obtain cells; Cells are seeded into the pancreatic cancer organoid culture medium according to any one of claims 1 to 2, and organoid growth culture is performed to obtain pancreatic cancer organoids.

4. The method for culturing pancreatic cancer organoids according to claim 3, characterized in that, The digestive fluid contains type 2 collagenase and neutral protease.

5. The method for culturing pancreatic cancer organoids according to claim 4, characterized in that, The final concentration of type 2 collagenase in the digestive fluid is 1-3 mg / ml; The final concentration of neutral protease in the digestive fluid is 0.01~0.03 U / ml; And / or, the digestion process takes 1 to 2 hours.

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